Two colchicine-binding domains of beta-tubulin have been identified by direct photolabeling, limited proteolysis with trypsin and chymotrypsin, immunoblot analysis, and sequencing of labeled peptides. The domains are (1) in the N-terminus starting at residue 1 and extending to residues 213/214 to 241/242. Thus, at least two domains of beta-tubulin are involved in colchicine binding. The structural consequence of colchicine binding has been probed by limited hydrolysis with trypsin or chymotrypsin of the colchicine-tubulin complex; this yields a different proteolytic pattern than free tubulin. A new cleavage site appears with both enzymes: at Lys392 for trypsin and Phe389 for chymotrypsin. This structural change in the carboxy-terminal portion of the beta-monomer may explain current models of substoichiometric inhibition of polymerization resulting from capping of the growing microtubule by a single layer of the colchicine-tubulin complex.